Background
Our project “The Gatekeeper” is based on two scientific publications : the work of Marciano et al. (1998) on the pIV porin electrophysiological characterisation, « An Aqueous Channel for Filamentous Phage Export. » and Spagnuolo et al.’s (2010) on defining the gating regions “Identification of the Gate Regions in the Primary Structure of the Secretin pIV.”
The pIV porin that we used for our project comes from the bacteriophage f1. It is a filamentous phage that infects Gram-negative bacteria and is capable of assembly and release without lysing the host cell, allowing it to continue to grow and divide. The assembly of the phage is coupled to its extrusion which is done by a machinery composed of three phage-encoded proteins : pI and pXI in the inner membrane and pIV in the outer membrane.
More precisely, pIV is secreted in the periplasm and then inserts into the outer membrane, where it forms a radially symmetrical 14-meric complex with a barrel like structure.
Marciano et al.(1998) brought a lot of elements to build our project and furthermore to create protocols. In fact, in this article, the pIV porin channel behavior (produced by the filamentous phage 1 to export virions out of its E. coli host) is analysed. This article describes two types of porin pIV, on the one hand the Wild Type porin and on the other hand a mutant porin pIV (pIV*S324G). Marciano and his team wanted to see if between those two pIV porins some differences in the permeability of the E. coli’s membrane appeared. Let’s remember that staple.
First thing to know, the phage F1 produces a channel porin pIV that is placed in the outer membrane of E. coli. This channel is closed (avoiding the lysis of the bacteria) except when the phage is leaving its host ⇒ So it’s possible to open this gate but surely thanks to a gating mechanism provided by the phage. However, this last article showed that when using the pIV mutant (pIV*S324G) the bacteria became more sensitive to the action of vancomycin. vancomycin is an antibiotic that can’t cross the outer membrane of bacteria because of its hydrophilicity. So if the bacteria expressing pIV proteins are sensitive to vancomycin it means that the channel pIV is opened. These effects are illustrated on the figure 1.
Figure 1: Graph showing the effect of vancomycin on the growth of E.Coli expressing or not pIV proteins or pIV* mutant proteins. IPTG is here used to induce the production of pIV via the control of the promoter of this gene. Marciano et al.
Figure 2: Primary structure of pIV. The “leaky” mutations are represented in green and are concentrated in GATE1 and GATE2. (Spagnuolo et al., 2010)
Beyond the use of vancomycin to highlight the permeability caused by the pIV*, Marciano et al, used a strain of E. coli with a deletion of the gene LamB. This one codes for a maltoporin placed in the outer membrane. Without this LamB gene, the bacteria can’t grow on maltodextrin (larger than triose) media; The concentration of maltodextrin was determined too (0.2% to 0.6%). Thanks to that method, bacteria with the open channel pIV and only them can grow on this media.
Finally this article estimated the diameter of the hole (6 nm) when the porin is open.
Thanks to all these precious informations contained in this article, we could optimize our selection method and several of our protocols.
Few years after, Spagnuolo et al.(2010) were able to identify residues in the pIV primary structure responsible for the gating mechanism. They achieved this by random mutagenesis of the pIV gene followed by selection for mutants with compromised barrier function, called “leaky”. They identified mutations in 34 residues, 30 of which clustered into two regions located in the center of the conserved C-terminal secretin family domain. They called these two regions GATE1 and GATE2, that span respectively 39 and 14 residues (figure 2). These informations allowed us to choose to target the regions GATE1 and GATE2 with our mutagenesis approach.
Bibliography
Marciano, Denise K., Marjorie Russel, et Sanford M. Simon. 1999. « An Aqueous Channel for Filamentous Phage Export ». Science 284 (5419): 1516‑19. doi:10.1126/science.284.5419.1516.
Spagnuolo, J., Opalka, N., Wen, W. X., Gagic, D., Chabaud, E., Bellini, P., Rakonjac, J. (2010). Identification of the gate regions in the primary structure of the secretin pIV. Molecular Microbiology, 76(1), 133–150. doi:10.1111/j.1365-2958.2010.07085.x
